A toolkit for expression of Strep-tagged enhanced green fluorescent protein concatemers in mammalian cells

Abstract

Green fluorescent protein (GFP) and its variants are widely used tools in life sciences. Recently, we and others have used enhanced green fluorescent protein (EGFP) concatemers for determination of nuclear localization signal strength, as natural fluorescence standards and for mapping mobility in living cell nuclei. In this study, we present a molecular toolbox of Strep-tagged EGFP concatemers ranging from 1 to 12 subunits (Addgene plasmids #122488-122499). EGFP concatemers can be easily fused to targeting motifs of any origin by oligonucleotide ligation. Subsequently, we used liposomal transfection for transient expression of EGFP concatemers in eukaryotic cells. We have tested multiple protocols for further processing of the cells and recommend use of formalin or paraformaldehyde/methanol fixation. After usage of these protocols, we were able to detect concatemers by both GFP fluorescence microscopy and αStrep immunomicroscopy. In addition, we observed a more reliable detection of the StrepTag polypeptide (SA-WSHPQFEK) when using αStrepTag antibody instead of StrepTag binding protein. Summing up, we present a toolbox for expression of a wide range of Strep-tagged EGFP concatemers for multiple applications. By use of EGFP fluorescence and/or StrepTag polypeptide, the expressed concatemers can be easily detected in the cell.

Keywords: Concatemer; EGFP; GFP; Multimer; Nuclear localization signal; StrepTag.